Abstract
It is well established that an independent inositide cycle is present within the nucleus, where it is involved in the control of cell proliferation and differentiation. Previous results have shown that when Swiss 3T3 cells are treated with insulin-like growth factor-I (IGF-I) a rapid and sustained increase in mass of diacylglycerol (DAG) occurs within the nuclei, accompanied by a decrease in the levels of both phosphatidylinositol 4-phosphate and phosphatidylinositol 4,5-bisphosphate. However, it is unclear whether or not other lipids could contribute to this prolonged rise in DAG levels. We now report that the IGF-I-dependent increase in nuclear DAG production can be inhibited by the specific phosphatidylinositol phospholipase C inhibitor 1-O-octadeyl-2-O-methyl-sn-glycero-3-phosphocholine or by neomycin sulfate but not by the purported phosphatidylcholine-phospholipase C specific inhibitor D609 or by inhibitors of phospholipase D-mediated DAG generation. Treatment of cells with 1-O-octadeyl-2-O-methyl-sn-glycero-3-phosphocholine or neomycin sulfate inhibited translocation of protein kinase C-alpha to the nucleus. Moreover, exposure of cells to 1-O-octadeyl-2-O-methyl-sn-glycero-3-phosphocholine, but not to D609, dramatically reduced the number of cells entering S-phase upon stimulation with IGF-I. These results suggest that the only phospholipase responsible for generation of nuclear DAG after IGF-I stimulation of 3T3 cells is PI-PLC. When this activity is inhibited, neither DAG rise is seen nor PKC-alpha translocation to the nucleus occurs. Furthermore, this PI-PLC activity appears to be essential for the G0/G1 to S-phase transition.
Highlights
These results suggest that the only phospholipase responsible for generation of nuclear DAG after insulin-like growth factor-I (IGF-I) stimulation of 3T3 cells is PI-PLC
We report that the IGF-I-dependent increase in nuclear DAG production can be inhibited by the specific phosphatidylinositol phospholipase C inhibitor 1-O-octadeyl2-O-methyl-sn-glycero-3-phosphocholine or by neomycin sulfate but not by the purported phosphatidylcholinephospholipase C specific inhibitor D609 or by inhibitors of phospholipase D-mediated DAG generation
Other investigations showed the existence of a relationship between mitogenic stimuli and changes in nuclear DAG mass: during compensatory hepatic growth, a peak in nuclear DAG levels was observed at 20 h following hepatectomy [19], and Sun et al [20] demonstrated that nuclear DAG rises at the G2/M transition sn-glycero-3-phosphocholine; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propane-sulfonate; NGS, normal goat serum; BSA, bovine serum albumin; PBS, phosphate-buffered saline; MES, 2-(N-morpholino)ethanesulfonic acid; CLSM, confocal laser scanning microscope
Summary
Materials—Dulbecco’s modified minimum essential medium, fetal calf serum, 1,2-dioleyl-3-palmitoyl-glycerol, dioleylglycerol, CHAPS, phosphatidylserine, neomycin, NGS, peroxidase-conjugated anti-rabbit IgG, Cy3-conjugated anti-mouse and anti-rabbit IgG, 5Ј-BrdUrd, histone H1, leupeptin, aprotinin, propranolol, phenylmethylsulfonyl fluoride, polyclonal antibody to PKC-␣, and BSA were from Sigma. Quiescent cultures were washed twice with serum-free medium containing 0.2% BSA and incubated in the same medium for the indicated times in the presence of 50 ng/ml of IGF-I. For experiments in the presence of inhibitors, isolated nuclei were preincubated with the chemicals for 5 min on ice. Detection of PKC-␣ by in Situ Immunofluorescence—Quiescent cultures of Swiss 3T3 cells were washed twice with serum-free medium containing 0.2% BSA and incubated in the same medium containing 50 ng/ml of IGF-I. In Vitro Assay for Nuclear PKC Activity—Isolated nuclei (10 g of protein) were incubated at 30 °C for 10 min in 20 mM Tris-Cl, pH 7.4, 10 mM MgCl2, 10 M ATP, 0.4 g/ml histone H1, 5 Ci of [␥-32P]ATP, in the presence of 1.2 mM CaCl2, 40 g/ml phosphatidylserine, and 3.3 M dioleylglycerol. All of the other differences were found to be not significant with p Ͼ 0.01
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